This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MscL, the mechanosensitive channel of large conductance, forms a homo-pentameric pore in which each subunit contains two transmembrane (TM) domains (TM1 and TM2) involved in the channel gating, and a C-terminal cytoplasmic helical domain. The crystal structure (PDB ID:2OAR) represents a closed state of the channel, showing a funnel shaped pore with a large opening on the periplasmic side and the narrowest point near the cytoplasm. Assuming that TM helix tilting due to membrane tension drives MscL gating, we will perform molecular dynamics simulations by applying the recently developed helix tilt restraint potential to the TM helices of MscL in a DMPC lipid bilayer. An initial structure of the MscL/DMPC complex without C-terminal helix (Gln110-Asn125) was generated using Membrane Builder in the CHARMM-GUI website (http://www.charmm-gui.org). CT?P (constant temperature, surface tension, and pressure) dynamics will be used to allow the system size along the XY axes to vary during the simulation. The P21 image transformation will be used to allow the number of lipid molecules in the top and bottom leaflets to vary during the simulations. Three different simulations will be performed by tilting (1) both TM1 and TM2 helices (10 restraints), (2) only TM1 helices (5 restraints), and (3) only TM2 helices (5 restraints). The tilt angles of TM1 helices will be gradually varied from 35 (PDB ID:2OAR) to 65 by 1 and those of TM2 from 33 (PDB ID:2OAR) to 63 by 1, together or separately. We will analyze the change of the pore size, molecular interactions, and pressure profile as a function of helix tilt to explore the possible gating mechanisms of MscL at the atomic level.